Many pathogenic bacteria express virulence genes that are required for infectivity and pathogenesis. Virulence factors are attractive targets for antibiotic discovery as there should be less selective pressure for the organisms to develop resistance since virulence factors are not essential for bacterial growth. However, targeting a large number of individual virulence factors makes this approach difficult. Expression of these genes is often controlled by specific virulence gene transcription factors of the AraC family. Inhibition of these transcription factors could represent a novel and general approach to antibiotic discovery. While much is known about the control of transcription in bacteria, those transcription factors involved in virulence are poorly understood. One example is Shigella flexneri, in which VirF, an AraC-type transcriptional regulator, is responsible for the expression of all downstream virulence factors that control intracellular invasion and cell-to- cell spread. Shigella infects ca. 165 million people and claims more than one million lives per year worldwide. While shigellosis has been considered to be a disease of the "third world", like many other highly contagious diseases it is rapidly spreading to developed countries. The emergence of drug and multi-drug resistant strains of Shigella emphasize the need for novel antibiotic development. Gene knock-out studies have validated that inhibition of VirF expression is sufficient to block the invasion cascade of Shigella in the host and thereby increase susceptibility to the host immune system. Our goals for this project are twofold. First, we wish to demonstrate the feasibility of targeting the regulation of virulence gene expression as a novel antibiotic approach using VirF as our model system. Second, the further development of validated hits against VirF will lead to novel agents against shigellosis, a desirable goal in itself as discussed above. The specific aims for this R21 application are two-fold. Aim 1: The development of assays for VirF, and adaptation of one or more assays for high throughput screening (HTS). HTS will be done on a chemically-diverse library of compounds (ca. 155,000 + 16,000 unique natural product extracts) held by the University of Michigan, Center for Chemical Genomics. Aim 2: "Hits" from HTS will be manually screened for potential "drugability" properties. Hits with appropriate properties will be further characterized via a number of in vitro assays. These assays are designed to validate the hits, provide guidance for subsequent lead optimization and also to probe mechanism(s) of inhibition. These studies will also help to elucidate the mechanism of action of VirF. In subsequent work, we will conduct a "hit-to-lead" campaign involving iterative cycles of analogue design, synthesis, and evaluation. We will also investigate the basic mechanisms of this poorly understood transcription factor. These studies will provide proof of principle for targeting virulence gene transcriptional activators with application to many human pathogens. This proposal has been revised to address the comments of the reviewers and to include progress that we have made on HTS assay development. PUBLIC HEALTH RELEVANCE: Relevance In the developing world, severe diarrhea is a killer, taking the lives of 2 to 3 million children every year. Persistent cases of diarrhea cause malnutrition, retard growth, and delay mental development. Shigella flexneri is one of the main causative agents of diarrheal disease. While shigellosis has been considered to be a disease of the "third world", like many other highly contagious diseases it is rapidly spreading to developed countries. Importantly, the emergence of a number of drug resistant strains of Shigella emphasizes the need for novel antibiotic development. This project will demonstrate the feasibility of targeting bacterial virulence- regulating transcription factors toward the discovery of novel antibiotics.